AU2021202603A1 - Novel therapy - Google Patents

Abstract

A method for treating cystitis, in particular acute cystitis, comprising administering to a patient in need thereof, an effective amount of a reagent selected from 5 the group consisting of IL- Iinhibitors and MMP inhibitors, or proteins selected from ASC or NLRP-3. Diagnostic methods are also described and claimed.

Description

Novel Therapy This application is a divisional of AU 2016205864, the entire contents of which are incorporated herein by reference. The present invention relates to methods for treating cystitis and in particular acute cystitis, to compositions for use in these therapies. Background of the Invention Urinary tract infections (UTIs) are common and may be dangerous. The clinical presentation and severity varies, depending on the site of infection and molecular basis of disease. In acute pyelonephritis (APN), bacteria ascend into the renal pelvis, where they cause an intense mucosal inflammatory response with progression into the renal parenchyma. Symptoms include high fever, malaise, loin pain as well as poor feeding and irritability in infants. In acute cystitis, infection is localized to the urinary bladder, resulting in dysuria, frequency and supra-pubic pain, typically without systemic involvement. While the clinical entities of acute pyelonephritis and acute cystitis typically are quite distinct, the molecular determinants of clinical presentation and severity are largely unknown. In acute pyelonephritis, a pathogen-specific TLR4 response is activated by P fimbriated F coli, through ceramide release and the successive phosphorylation of the TICAM-1 (TRIF) and TICAM-2 (TRAM) adaptors, CREB-1, c-FOS and c-JUN activates IRF- and API- dependent transcription. Additional involvement of MyD88, TIRAP and NF-kB depends on the virulence repertoire of the infecting strain. Genetic studies in the murine UTI model have identified IRF3-dependent gene expression and mCXCR2 dependent neutrophil activation as determinants of bacterial clearance and tissue homeostasis. Infected IrJ3+ or mCxcr2+ mice develop severe APN and tissue damage after one week and relevance for human APN susceptibility has been demonstrated, through disease-associated IRF3 and CXCR1 polymorphisms in APN prone patients. The cause of acute cystitis susceptibility has not been defined, however and it is rapidly becoming a therapeutic enigma as antibiotic resistance is reducing the options for treatment to a minimum. Genetic markers of APN susceptibility show no association to acute cystitis, emphasizing the differences in pathogenesis and genetic control. Acute uncomplicated cystitis is characterized by localized inflammation and symptoms from the lower urinary tract, typically dysuria and urgency and frequency of urination. It occurs predominantly in girls and healthy women with normal urinary tracts and at least 60% of all females will report an episode during their lifetime. The recurrence rate is high, especially in a subset of patients, where recurrent cystitis episodes may cause chronic tissue damage and impact the quality of life. In addition, acute cystitis patents pose a highly significant challenge to the health care system.

The urinary bladder mucosa is often under microbial attack but does not always

retaliate with such force, as in acute cystitis. In asymptomatic carriers, the epithelium

remains fairly unresponsive, despite bacterial numbers well above 101 CFU/ml (9, 10). It

is therefore challenging to understand, at the molecular level, how a state of exaggerated

inflammation can be generated specifically in acute cystitis patients. Interactions of

bladder cells with pathogenic bacteria have been shown to trigger an innate immune

response, involving the epithelium and adjacent mucosal cells, such as mast cells and

macrophages. Uroepithelial cells from women with recurrent cystitis have an increased

density of receptors for adhering bacteria and bacterial persistence in intracellular

communities has been studied as a reservoir of infection, but the molecular basis of

susceptibility and disease has remained unclear.

Summary of the Invention

The applicants have now identified for the first time, a cellular and genetic basis

for acute cystitis susceptibility. In a screen for cytokines produced by infected human

bladder cells, they discovered that about 85% of epidemiologically defined acute cystitis

E. coli strains triggered the secretion of IL-P, a classical pro-inflammatory cytokine,

which drives the pathogenesis of human inflammatory disorders. To address if the

inflammasome controls host susceptibility to acute cystitis, bladder infection was

established in mice lacking IL-lb, ASC (apoptosis-associated speck-like protein

containing a CARD) or NLRP-3 (NACHT, LRR and PYD domains-containing protein 3) also known by cryopyrin). Consistent with such a role,Il-1b` mice were protected from

pathology, as were Casp14 mice which have an IL-Ip secretion deficiency. Remarkably,

Asc' and Nlrp3k mice, which are deficient for inflammasome constituents, developed an

IL-Ip hyperactivation state with rapid, fulminant bladder inflammation and tissue

damage, accompanied by massive neutrophil infiltration and upregulation of

inflammasome genes. Mutations in these ASC or NLRP-3 genes in humans may therefore provide an indicator of susceptibility to disease, and also provide a prophylactic or therapeutic strategy for addressing disease.

The applicants have also identified a new mechanism of IL-Ip processing which

is responsible for this phenotype, involving the metalloproteinase MMP-7 in MMP-7

dependent processing and MMP-7 overexpression, in the bladder mucosa. The MMP-7

response may be explained by a direct effect of ASC and NLPR-3 on theMMP7

promoter, resulting in de-repression of Mmp7 expression in Asc-- and Nlrp3-/-mice. As a

consequence, the applicants found that treatment of Asc-/- mice by immunomodulation

with the IL-1 receptor antagonist (IL-iRA) Anakinra or a metalloproteinase inhibitor

prevented acute cystitis and pathology and Illb-/mice were protected. The results

identify acute cystitis as a hyper-inflammatory condition caused by IL-p

hyperactivation and inflammasome dysregulation. It may be related to other IL-ip

driven hyper-inflammatory disorders.

The present invention provides a method for treating cystitis comprising

administering to a patient in need thereof, an effective amount of a reagent selected from

the group consisting of IL-ip inhibitors and MMP inhibitors.

A method according to claim 1 wherein the cystitis is acute cystitis.

In particular, the reagent is an IL-Ip inhibitor, and many such compounds are

known in the art. These include for example, small molecules such as anthraquinones,

described for example in USP 4,244,968 including diacerein, as well as proteins and

peptides such as interleukin-1 receptor antagonist (IL-1 RA) for example anakinra, and

rilonacept, or pharmaceutically acceptable salts thereof, or prodrugs thereof, and

combinations of these. In particular, the reagent is an IL-p receptor antagonist, such as

anakinra (US Patent No 5,075,222).

Alternatively, the reagent is an MMP inhibitor, and in particular an MMP7

inhibitor. A wide range of MMP inhibitors are known as described for example

Durrant et al. Chem. Biol. Drug Des 20111; 78; 191-198, the content of which is

incorporated herein by reference. Particular examples include batimastat, periostat

(doxycycline hyclate), marimastat, or salts or prodrugs thereof, but in particular

batimastat.

The invention further provides a reagent selected from the group consisting of

IL-Ip inhibitors and MMP inhibitors for use in the treatment of cystitis, in particular

acute cystitis. Particular reagents are as described above.

The applicants have found that in patients suffering from cystitis have elevated

levels of IL-Ip and MMP-7 in urine, and thus these molecules act as diagnostic

biomarkers. Hence, the invention further provides a method for diagnosing cystitis and

in particular, acute cystitis, said method comprising detecting elevated levels of IL-ip or

MMP-7 in urine of a subject. Suitable detection methods are known in the art, and

include ELISA.

In yet a further aspect, the invention provides a method for diagnosing

susceptibility to cystitis, said method comprising detecting a mutation in a gene

encoding a protein selected from ASC or NLRP-3 which results in downregulation of

said gene and/or in the expression of inactive protein. As the applicants have

demonstrated, absence of these proteins leads to increased susceptibility to cystitis.

Without being bound by theory, this may be due to the suppression of MMP-7

expression by these proteins, as discussed further below. Therefore, patients having a

mutation which impacts on the expression of these proteins or the expression of

functional proteins are likely to be at risk of developing cystitis as a result of elevation of

MMP-7 levels.

Diagnosis may be carried out at the gene level, whereby the sequence of the Asc

and/or Nlrp-3 is completely or partially determined and compared with a normal gene.

For example, sites of common mutations that leads to inactivation or downregulation of

the ASC or NLRP-3 proteins may be analyzed and the presence or absence of the

selected mutation may be used to assess the likelihood that the patient is susceptible to

cystitis.

Alternatively, diagnosis may be carried out at the protein level, where a suitable

sample from a subject, such as a blood, serum, plasma or urine sample is analyzed for

the presence of active ASC or NLRP-3 protein. Suitable methods in this case may

include immunochemical assays such as ELISAs which use antibodies specific for the

target proteins.

Once diagnosed, administration to the subject of the proteins or functional

equivalents thereof, would be expected to prevent or treat disease. Thus in yet a further

aspect, the invention provides a method for preventing or treating cystitis in a patient

susceptible thereto as a result of a mutation which impacts on the expression of

functional ASC or NLRP-3, which method comprises administering to said patient a

protein selected from ASC or NLRP-3 or a functional fragment or variant thereof.

As used herein, the expression 'fragment' refers to a peptide or protein which

lacks one or more amino acids found in a full length protein but which still has the

function of the full length protein.

As used herein, the expression 'variant' refers to a peptide sequence in which the

amino acid sequence differs from the basic protein or peptide sequence in that one or

more amino acids within the sequence are substituted for other amino acids. However,

the variant produces a biological effect which is similar to that of the basic sequence.

Amino acid substitutions may be regarded as "conservative" where an amino

acid is replaced with a different amino acid in the same class with broadly similar

properties. Non-conservative substitutions are where amino acids are replaced with

amino acids of a different type or class.

Amino acid classes are defined as follows:

Class Amino acid examples

Nonpolar: A, V, L, I, P, M, F, W

Uncharged polar: G, S, T, C, Y, N, Q Acidic: D, E

Basic: K, R, H.

As is well known to those skilled in the art, altering the primary structure of a

peptide by a conservative substitution may not significantly alter the activity of that

peptide because the side-chain of the amino acid which is inserted into the sequence may

be able to form similar bonds and contacts as the side chain of the amino acid which has

been substituted out. This is so even when the substitution is in a region which is critical

in determining the peptide's conformation.

Non-conservative substitutions may also be possible provided that these do not

interrupt the function of the protein or peptide.

Broadly speaking, fewer non-conservative substitutions will be possible without

altering the biological activity of the polypeptides.

In general, variants will have amino acid sequences that will be at least 70%, for

instance at least 71%, 75%, 79%, 81%, 84%, 87%, 90%, 93% or 96% identical to the basic

sequence. Identity in this context may be determined using the BLASTP computer

program with the basic native protein sequence as the base sequence. The BLAST

software is publicly available at http://blast.ncbi.nm.nih.gov/Blast.cgi (accessible on 12

March 2009).

Variants may also include addition sequences such as tag sequences that may be

used for instance in facilitating purification of the peptide or in detection of it. Thus for

instance, the variant may further comprise an affinity tag such as chitin binding protein

(CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), FLAG, myc,

biotin or a poly(His) tag as are known in the art. In another embodiment, the variant

may comprise a fluorescent protein such as green fluorescent protein (GFP).

In a further aspect, the invention provides a protein selected from ASC or NLRP

3 or a functional fragment or variant thereof, for use in the treatment of patients

suffering from or susceptible to cystitis as a result of a mutation which impacts on the

expression of functional ASC or NLRP-3 respectively.

For administration to patients, the reagent or protein is suitably administered in

the form of a pharmaceutical composition, which further comprise a pharmaceutically

acceptable carrier. Such compositions are known in the art.

Suitable pharmaceutical compositions will be in either solid or liquid form. They

may be adapted for administration by any convenient route, such as parenteral, oral or

topical administration or for administration by inhalation or insufflation. The

pharmaceutical acceptable carrier may include diluents or excipients which are

physiologically tolerable and compatible with the active ingredient.

Parenteral compositions are prepared for injection, for example either

subcutaneously or intravenously. They may be liquid solutions or suspensions, or they

may be in the form of a solid that is suitable for solution in, or suspension in, liquid prior

to injection. Suitable diluents and excipients are, for example, water, saline, dextrose,

glycerol, or the like, and combinations thereof. In addition, if desired the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH-buffering agents, and the like.

Oral formulations will be in the form of solids or liquids, and may be solutions,

syrups, suspensions, tablets, pills, capsules, sustained-release formulations, or powders.

Oral formulations include such normally employed excipients as, for example,

pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium

saccharin, cellulose, magnesium carbonate, and the like.

Topical formulations will generally take the form of suppositories or intranasal

aerosols. For suppositories, traditional binders and excipients may include, for example,

polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures

containing the active ingredient.

The amount of reagent administered will vary depending upon factors such as

the nature of the reagent being used, the size and health of the patient, the nature of the

condition being treated etc. in accordance with normal clinical practice. Typically, a

dosage in the range of from1 tg-50mg/Kg for instance from 2-20 mg/Kg, such as from 5

15 mg/Kg would be expected to produce a suitable effect.

The urinary bladder mucosa is often under microbial attack but rarely retaliates

with full force. In asymptomatic carriers, the epithelium remains refractory despite

bacterial numbers well above 101 CFU/ml. Yet bacteria that cause acute cystitis create a

state of exaggerated inflammation and the patients develop the characteristic symptoms

of dysuria, frequency and pain. In this study, we elucidate molecular and cellular

mechanisms underlying the exaggerated response that leads to acute symptoms and

bladder pathology. We identify IL-lb as a crucial host sensor of bladder infection and

inflammasome dysregulation as a cause of bladder pathology. In addition, the

metalloproteinase MMP-7 is introduced as a new molecular player in mucosal

inflammation, proteolytically cleaving IL-lb. The importance of IL-lb and MMP-7 is

proven by treatment of susceptible mice with IL-IRA or MMP inhibitor. Elevated IL-lb

levels were also detected in patients with acute cystitis compared to patients with ABU.

This molecular basis of acute cystitis also provides as a new therapeutic approach using

IL-lb and MMP-7 inhibitors as immune-modulators of acute cystitis.

IL-lb is a potent pro-inflammatory cytokine that initiates and amplifies innate

immune responses. IL-lb production increases in response to viral, bacterial, fungal and

parasitic infections and IL-lb is essential for the defense against microbial attack. IL-lb

responses may also be detrimental, however, and dysregulation of IL-lb has been

observed in autoimmune and auto-inflammatory disorders, such as rheumatoid arthritis,

multiple sclerosis, Crohn's disease or neuro-degenerative disorders. This dichotomy was

apparent also in the present study, where a controlled IL-lb response accompanied the

clearance of infection in WT mice. The association of a dysregulated IL-lb response with

disease suggested that acute cystitis is an infection-induced, hyper-inflammatory

disorder of the urinary bladder. This is in contrast to Salmonella and Shigella, where Illb

mice showed increased mortality rather than protection. The dramatic phenotype in Asc

' and Nlrp3+ mice raises the question if ASC and NLRP-3 dysfunctions may precipitate

other hyper-inflammatory conditions, where pathology is associated with IL-lb and

inflammasome responses.

The NLRP-3 inflammasome is activated by a number of microbial stimuli

including LPS, MDP, bacterial RNA, poly(I:C) as well as ATP and bacterial pore-forming

toxins. NLRP-3 activation stimulates the binding of ASC through homotypic pyrin

domains and the caspase recruitment domain of ASC serves as an adaptor that binds

NLRP-3 to pro-Caspase-1 to form the inflammasome. The dramatic disease phenotype in

Asc' and Nlrp3' mice strongly suggested that a functional inflammasome response is

required to maintain tissue homeostasis in infected bladders. Cystitis strains that were

active IL-lb inducers also increased the expression of NLRP-3, ASC and Caspase-1 in

human bladder cells and processing of IL-lb was detected, with secretion of the mature

form into urine and cell supernatants. Furthermore, the presence of large quantities of

IL-lb in the urine of Asc' and Nlrp3 mice demonstrated that pro-IL-lb is processed in

these mice. Yet, effects of caspase inhibition were limited, suggesting that additional

mechanisms must be involved.

Proteolytic cleavage by MMP-7 was identified as a new mechanism of IL-lb

processing and evidence for IL-lb fragmentation by MMP-7 was obtained by direct

cleavage of the purified components, in vitro. MMP-7 has metalloendopeptidase activity

and is known to degrade collagen, proteoglycans, fibronectin, elastin and casein.MMP7 is activated following TNF-a or IL-lb stimulation of cells. It is commonly expressed in epithelial cells and has been shown to regulate the activity of defensins in the intestinal mucosa. By transcriptomic analysis, Mmp7 was identified as the most strongly activated gene in pathological bladders and the protein was mainly expressed in the bladder epithelium. The dramatic shedding of epithelial cells positive for IL-p and MMP-7 may, thus, explain the elevated urine IL-lb levels in mice that developed pathology and the low IL-lb levels in mice treated with the MMP inhibitor. In the absence of inhibitor, this process resulted in a denuded mucosal surface, lacking a protective epithelial barrier.

These morphological changes make it possible to understand the intense pain and

symptoms in acute cystitis patients.

The results also emphasize the difference in pathogenesis between acute cystitis

and acute pyelonephritis. The pathology in Asc+ and Nlrp3+mice was bladder specific as

there was no evidence of exaggerated inflammation in the kidneys. Conversely, Irf3+

mice, which develop massive kidney pathology in response to CFT073 infection, showed

no bladder pathology. This discrepancy emphasizes that bladder pathology is generated

by mechanisms that differ from the well-known molecular interactions and signaling

pathways that control acute pyelonephritis. Interestingly, this includes a difference in

TLR4 dependence. Kidney pathology is controlled, to a large extent by TLR4 (7, 8), acting

upstream of the IRF-3 and AP-1 transcription factors. Kidneys of Tlr4mice therefore fail

to respond to most UPEC strains and develop ABU. In contrast, a residual response was

present in the bladders of Tlr4mice, through MyD88 and NF-kB signaling, Illb and Tnf

expression, potentially suggesting a mechanism for the increased expression of IL-lb and

IL-lb-dependent genes.

Recurrent acute cystitis is a handicap, socially, professionally and emotionally

but despite its prevalence and importance for patients and society, acute cystitis is a

poorly understood disease. Social and behavioral factors have been emphasized as a

cause of recurrent infections and until recently, therapeutic options have included a

variety of shorter or longer antibiotic regimens, many of which have been discontinued,

due to resistance development. It comes as no surprise, that this highly painful condition

has been the focus of various interventions in addition to antibiotic therapy. Deliberate

establishment of competitive microflora has shown promising clinical effects, but novel, therapeutic approaches are needed in this large patient group. In this study, we show that acute cystitis is amenable to IL-lb receptor blockade in mice, given before infection and daily during the 7-day experiment. Inhibition of MMP-7 was also protective, but with a less complete phenotype than mice receiving the IL-IRA. As IL-IRA is in clinical use, testing immunotherapy with drugs like Anakinra as an adjunct to antibiotics may be a realistic option in acute cystitis patients.

FIGURE LEGENDS

Figure 1. Acute cystitis strains activate IL-1p and inflammasome responses in human

bladder epithelial cells.

(A) IL-Ip response in human bladder epithelial carcinoma cells (HTB-9) infected with

acute cystitis strains CY-92, CY-17, CY-132 and CY-49 (108 CFU/ml, 4 hours). CFT073

and asymptomatic carrier strain E. coli 83972 (ABU) were used as controls. IL-Ip was

quantified by ELISA, in cell supernatants (n = 3, means ±SEM, * P < 0.05, two-tailed

Mann Whitney test). (B) Increased pro-IL-1p and mature IL-ip levels in cells infected

with CY-92, CY-17 and CY-132 (Western blot analysis of cell supernatants). (C) IL-1p

response to an epidemiologically defined collection of paediatric acute cystitis strains (n

= 67) compared to ABU strains (n = 66),obtained from children in the same geographic

area. Pie chart indicating the frequency of bacterial strains activating IL-p responses:

high (orange), intermediate (blue), low (purple) or negative (green). Histogram (inset) of

the mean IL-Ip response to CY versus ABU strains (means ±SEM, *** P < 0.001, two tailed Mann Whitney test). (D) Increased IL-Ip and inflammasome protein staining

(NLRP-3, ASC and Caspase-1) in human bladder cells infected with CY-17, CY-92, CY

132, CFT073 or ABU. Scale bar = 20 tm.

Fig 2. Bladder pathology in infected Asc-/- and Nlrp3-/- mice.

(A) Macroscopic bladder inflammation in WT mice infected with CY-17, CY-92 or

CFT073 for seven days (left panels). Moderate edema and hyperaemia with a loss of

structure in H&E-stained tissue sections (middle panels). Staining for bacteria and

neutrophils, by immunohistochemistry (right panels). Scale bars = 1 mm (whole bladders), 100 tm (H&E) and 50 tm (immunofluorescence). (B) Pathology scores (left

panels). Kinetics of infection and neutrophil recruitment (n = 4 per group, means ±SEMs,

** P < 0.01, * P < 0.05, two-tailed unpaired t-test, compared to CFT073 infection). (C)

Tissue response to infection in Asc-/- and Nlrp3-/- mice. Elevated bacterial counts in

bladder and urine (upper panels) and neutrophil counts, compared to WT and Il b-/

mice (n = 10-14 per group, means ±SEMs of two experiments, *** P < 0.001, ** P < 0.01, * P

< 0.05, two-tailed unpaired t-test compared to WT mice). (D) Macroscopic bladder

pathology (left panels) in Asc-/- and Nlrp3-/- mice, 7 days after infection. Loss of

structure in H&E-stained tissue sections (middle panels). Immunohistochemistry (right

panels), showing bacterial/neutrophil infiltrates and micro abscesses along the mucosal

border. Illb-I- mice were protected against inflammation, with intact bladder

morphology (H&E), few bacteria and low neutrophil counts (immunohistochemistry).

Scale bars = 1 mm (whole bladders), 200 tm (H&E) and 50 m (immunofluorescence).

Fig 3. Hyper-activation of IL-1p dependent gene expression in Asc-/- and Nlrp3-/

mice.

Reprogramming of host gene expression by infection, defined by transcriptomic analysis

of whole bladder RNA from infected mice (7 days) compared to uninfected controls of

each genotype (FC 1.41, P < 0.05). (A) A distinct gene set distinguished the Asc-/- and

Nlrp3-- mice bladders with acute cystitis from mice without pathology (Heat map of differentially expressed genes). (B) Histogram quantifying the genotype-specific

response to infection. From 3,000-6,000 genes were regulated in Asc-/- and Nlrp3-- mice

that developed acute cystitis compared to 300-2,000 genes in Illb-I- and WT mice (P =

0.014, unpaired t-test). (C) About 1800 genes regulated by infection in mouse bladders

were shared among the mice that developed acute cystitis. (D) Massive over-expression

of IL-1p and genes encoding inflammasome activators or downstream effectors in Asc-/

and Nlrp3-- mice with acute cystitis but not in Ilb-- and WT mice.

Fig 4. IL-1 processing by MMP-7.

(A) Gene expression profiling of whole bladder mRNA identified Mmp7 as the top up

regulated gene in Asc-/- and Nlrp3-/- mice with bladder pathology (CFT073 infected

mice, 7 days). (B) Mmp7 expression quantified by qRT-PCR (n = 2 mice per group, mean

±SEMs). (C) Epithelial MMP-7 staining in Asc-- and Nlrp3-/- mice with bladder

pathology. Scale bars = 50 tm. (D) Proteolytic cleavage of Il-1p by MMP-7 in vitro, using

purified enzyme and GST-tagged IL-1p. Arrows indicate cleavage products. The

proteolytic IL-1p fragments were of similar size as the bands detected in supernatants of bladder cells infected with CY-92. (E) Lack of pathology in Mmp7-/- mice, 7 days after infection with CFT073. Low bacterial and neutrophil counts in urine compared to Asc-/ mice (n = 5 per group, means ±SEMs, ** P < 0.01, two-tailed unpaired t-test). Scale bar = 1 mm (whole bladders), 100 tm (H&E) and 50 tm (immunofluorescence). (F)

Elevated IL-1p secretion into the urine of Asc-/- mice, but not CaspI-/- or Mmp7-/- mice.

Limited retention of IL-1p in bladder tissue of Mmp7-/- mice, 7 days after infection. Scale

bar = 50 tm. Fig 5. Regulation of MMP7 expression by ASC and NLRP-3.

(A) MMP-7 response to infection with CY-17, visualized 739 by confocal microscopy.

Massive increase in MMP-7 staining after transfection with ASC or NLRP3 specific

siRNAs (left panels, scale bar = 20 tm). (B) Western blot, confirming the knock-down of

ASC or NLRP-3. (C) PCR amplification of a 259 bp fragment in theMMP7 promoter (P1,

743 -18/-276 relative to transcription start site). (D) EMSA of a nuclear extract from CY-17

infected HTB-9 cells and P1. Binding identified as a band shift (arrow), was inhibited by

ASC- or NLRP-3 specific antibodies. Free DNA forms a single low molecular weight

band. (E) EMSA of the 259 bp MMP7 promoter fragment P1 and recombinant ASC. The

dose-dependent formation of an ASC-P1 complex is shown as a band shift, which was

inhibited by anti-ASC antibodies. IgG was used as negative control. Negative control

proteins (murine IgG, bovine actin and MMP-7) did not cause a band shift.

Fig 6. Acute cystitis immunotherapy, using an IL-I receptor antagonist (IL-iRA) or an

MMP inhibitor.

(A) Anakinra (IL-iRA) protects susceptible Asc-/- mice from acute cystitis. The mice were

pre-treated with Anakinra, 30 min before infection and daily after infection with E. coli

CFT073 (1mg in 100tl of PBS i.p. per mouse) Alternatively, Asc` mice were pre-treated

with the matrix metalloproteinase inhibitor (MMPi) Batimastat, 30 minutes before

infection and daily after infection with E.coli CFT073 (0.5mg in 100tl of PBS i.p. per

mouse, except day 3). (B) IL-iRA prevented macroscopic bladder pathology in Asc+

mice, as did the MMPi. Scale bar = 1mm (C) The disease severity score was reduced by

the inhibitors (n=3-4, ***P<0.001, compared to untreated Asc- mice, Fisher's exact test).

(D) Protection from bladder tissue pathology in treated mice (H&E). Arrows indicate mucosal sloughing, edema and subepithelial abscesses in untreated mice. Loss of neutrophil aggregation in bladder sections from treated mice. Scale bar = 200 tm

(H&E) and 50 tm (immunofluorescent staining). (E) Neutrophils and bacteria in the

urine of IL-IRA or MMPI treated Asc-/- mice, compared to untreated mice, 7 days after

infection with CFT073 (n = 3-4, means ±SEM, ** P < 0.01, * P < 0.05, two-tailed unpaired t

test). Fig 7. Elevated concentrations of IL-11 and MMP-7 in the urine of patients with acute

cystitis.

(A) IL-1p concentrations were higher in urine samples from patients with acute cystitis

(n = 9) than in patients with ABU, who were asymptomatic carriers of E. coli 83972 (n=

31). Histogram (inset) compares mean IL-1p concentrations between the two patient

groups (mean ±SEM, *** P < 0.001, two-tailed Mann Whitney test). (B)MMP-7

concentrations were higher in urine samples from patients with acute cystitis than in

patients with long-term ABU (*** P < 0.001, two-tailed Mann Whitney test). Urine

samples were obtained at the time of diagnosis from patients with sporadic acute cystitis

and from patients with ABU, who carried E. coli 83972 after therapeutic inoculation (9). Detailed Description of the Invention

Example 1

Methods and Materials

Patients with acute cystitis were enrolled at two primary care clinics. A diagnosis

of acute cystitis was based on a urine dipstick analysis positive for bacteria and

symptoms from the lower urinary tract, including frequency, dysuria and suprapubic

pain. Midstream urine specimens were obtained at the time of diagnosis.

Patients with ABU were included in a placebo-controlled study of intravesical

inoculation with E. coli 83972 ( Sunden F, et al. TheJournalof urology. 2010;184(1):179-85).

Briefly, E. coli 83972 bacteriuria was established by intravesical inoculation (101 CFU/ml

in saline), daily for three days and the outcome was measured as the total number of

UTIs during an optimal period of 12 months followed by a cross over to a similar period

without E. coli 83972 bacteriuria. Urine samples were obtained for cytokine analysis

during E. coli 83972 bacteriuria and symptom scores were registered. Urine IL-1p or

MMP-7 levels were quantified by ELISA with Human Il-1b/IL-1F2 DuoSet ELISA kit and

Human total MMP-7 Immunoassay Quantikine@ ELISA respectively (both from R&D

Systems).

Bacterialstrains

Cystitis (CY) and asymptomatic bacteriuria (ABU) strains were prospectively

isolated during a study of childhood UTI in Gteborg, Sweden (Lindberg U, et al. The

Journalofpediatrics. 1978;92(2):194-9). The UPEC strain, E. coli CFT073 (06:K2:H1)

(Nielubowicz GRet al. Nature reviews Urology. 2010;7(8):430-41), and ABU strain E. coli

83972 (OR:K5:H-) were controls. Bacteria were cultured on tryptic soy agar (TSA, 16 h,

37°C), harvested in phosphate-buffered saline (PBS, pH 7.2) and diluted as appropriate.

Overnight static cultures of E. coli CFT073 in Luria-Bertani (LB) broth were used for

experimental infection.

Cell culture Human bladder (5637, ATCC# HTB-9) and kidney epithelial cells (A498, ATCC#

HTB-44) were cultured to 70-80% confluence on 8-well chamber slides (6x10 4 cells/well)

or in 6-well plates (6x101 cells/well) in RPMI-1640 (Thermo Scientific) supplemented with

1 mM sodium pyruvate, 1 mM non-essential amino acids, gentamicin (50 tg/ml) (GE

Healthcare) and 10% heat-inactivated fetal bovine serum (PAA) at 37°C with 5%CO2.

Cytokine measurements

IL-1p, IL-6 and IL-8 concentrations in filtered supernatants (Syringe Filter w/0.2

um PES, VWR) were determined by Immulite 1000 (Siemens) and l-1P concentrations in

cell supernatants or urine by Human or MouseIl-1b/IL-F2 DuoSet ELISA kits (R&D

Systems). Urine MMP-7 levels were quantified with Human total MMP-7 Immunoassay

Quantikine(RTM)ELISA (R&D Systems).

Confocal microscopy Cells were grown as described above, infected, fixed (3.7% formaldehyde, 10

min), permeabilized (0.25% Triton X-100, 5% FBS, 15 min), blocked (5% FBS, hour at

room temperature), incubated with primary antibodies in 5% FBS overnight at 4°C

(rabbit anti-IL-i beta (ab9722), anti-TMSI (ASC) (ab155970), anti-caspase-i (ab62399, all

Abcam) or mouse anti-CIASI/NLRP3 (ab17267, Abcam) and appropriate fluorescently

labeled secondary antibody (Alexa Fluor@ 488 goat anti-rabbit IgG (#A-11034) and goat

Anti-Mouse IgM (#A-11001, both Life Technologies), hourr at room temperature).

After nuclear staining (DRAQ5, Abcam), slides were mounted (Fluoromount, Sigma

Aldrich), imaged by laser-scanning confocal microscopy (LSM510 META confocal

microscope, Nikon Eclipse Cl) and quantified by ImageJ software 1.46r (NIH).

Western blotting Cells were cultured as described above, but in particular in 6-well plates (7x10 5

cells/well, Thermo Fisher Scientific), lysed with RIPA lysis buffer, supplemented with

protease and phosphatase inhibitors (Roche Diagnostics). They may then be fractionated

using NE-PER Nuclear and Cytoplasmic extraction reagents (Thermo Scientific). For

Caspase inhibition, cells were pre-incubated with Z-VAD(OMe)-FMK (#BML-P416-0001,

Enzo Life Sience, 100 tM, 30 min). Supernatants were filtered and concentrated by

trichloroacetic acid precipitation, followed by acetone desiccation. Lysates and

supernatants were run on SDS-PAGE (4-12% Bis-Tris gels, Invitrogen), blotted onto PVDF membranes which were blocked with 5% bovine serum albumin (BSA) or non-fat

dry milk (NFDM) and incubated with primary antibody: rabbit anti-IL-i beta (1:2 000, or

1:2,500 in 5%NFDM, ab9722), rabbit anti-ASC (1:200 in 5% BSA, sc-22514-R, Santa Cruz),

mouse anti-390 NLRP3/NALP3 (1:400 in 5% BSA, Cryo-2, Adipogen) or rabbit- anti

MMP-7 (1:1000, ab4044, both Abcam), washed with PBS tween 0.1%, followed by

incubation with secondary antibodies in 5% NFDM (goat anti-rabbit IgG-HRP or goat

anti-mouse HRP (Cell Signaling). Bands were imaged using ECL Plus Western Blotting

Reagent (GE Healthcare) with HRP-linked anti-GAPDH (1:1000, sc-25778, Santa Cruz)

as loading control. Restore Western Blot Stripping Buffer (Pierce) was used as indicated.

Bands were quantified by ImageJ.

Co-Immunoprecipitation

Nuclear fractions, extracted as previously described, were incubated with rabbit

anti-ASC antibody (sc-22514-R, magnetic Dynabeads@ Protein G (Life technologies),

analyzed by SDS-PAGE with rabbit anti- ASC and mouse anti-NLRP3 (Cryo-2,

Adipogen) primary antibodies (1:200-1:1000, 5 %BSA), followed by secondary anti

rabbit (Cell Signaling) or anti-mouse (DAKO) antibodies (1:4000, 5% NFDM).

Globalgene expression

Total RNA was extracted from murine bladders in RLT buffer with 1%3

Mercaptoethanol after disruption in a tissue homogenizer (TissueLyser LT, Qiagen) using Precellys@ Lysing kits (Bertin Technologies), with the RNeasy@ Mini Kit (Qiagen), amplified using GeneChip 3'IVT Express Kit, hybridized onto Mouse Genome 430 PM array strips for 16 hours at 45°C, washed, stained and scanned using the Geneatlas system (all Affymetrix).

Transcriptomic data was normalized using Robust Multi Average implemented

in the Partek Express Software (Partek). Fold change was calculated by comparing

infected to uninfected mice of the same genetic background (cut off fold change > 1.41).

Heat-maps were constructed by Gitools 2.1.1 software. Differentially expressed genes

and regulated pathways were analyzed by Ingenuity Pathway Analysis software (IPA,

Ingenuity Systems, Qiagen). Qiagen's list of 84 key inflammasome genes was selected for

analysis.

QuantitativeRT-PCR

Complementary DNA was reverse-transcribed from 1 tg of mice total RNA

using SuperScriptTM III First-Strand Synthesis System for RT-PCR (Invitrogen), and

quantified in real time using qSTAR qPCR primer pairs against Mus musculus Illb and

Mmp7 genes (#MP206724 and #MP207902, Origene) and QuantiTect@ SYBR@ Green PCR

kits (Qiagen) on a Rotor Gene Q (Qiagen). qRT-PCR reactions were run in technical

duplicates and gene expression was analyzed based on a standard curve for each primer

pair.

In vitro proteolysis

Recombinant human IL-1 P NLRP-3 or PYCARD (ASC) (280 ng, H00003553-PO2,

H00114548-P01 and H00029108-P01 respectively, Abnova) were incubated with

recombinant active human MMP-7 (0.035U, #444270 Merck Millipore) in MMP reaction

buffer (20 mM Tris, pH 7.6, 5 mM CaC12,0.1M NaCl) at 37°C until stopped with 100 mM

DDT. Fragments were detected by Western blot, using rabbit anti-IL-1 beta (1:2 000,

ab9722, Abcam), rabbit anti-TMS1 (ASC) (1:1000, ab155970 or p9522-75, US Biological)

and mouse anti-CIAS1/NLRP3 (1:500, ab17267, Abcam) or rabbit anti-NLRP3 (1:500, sc

66846, Santa Cruz). siRNA transfection

HTB-9 cells were transfected with PYCARD/ASC and NLRP3 specific siRNAs

(0.09 tM,FlexiTube GeneSolution, #GS29108 and #GS114548, Qiagen) or with AllStars

Negative Control siRNA (#SI03650318, Qiagen) using the HiPerFect Transfection

Reagent (#301705, Qiagen) for 17 hours, then infected. Transfection efficiency was

assessed by Western blotting (Fig 5B).

PCR analysis

MMP7 promoter and promoter flanks were amplified in 10 different fragments

by PCR using 15 ng of total human genomic DNA and suitable forward and reverse

primers (http://primer3.ut.ee/). Thermal cycling conditions were as follows: 95°C for 2

min, 35 cycles (95°C for 30 s, 60°C for 30 s and 72°C for 40 s) and 72°C for 5 min. Electrophoretic mobility shift assay (EMSA)

Amplified DNA sequences from the MMP7 promoter were used as probes and

labelled with GelGreen (Biotium). Each reaction contained 3-5 tg of DNA probe with,

5tg of nuclear extract from infected HTB-9 cells, or 0.2-0.65 tg recombinant ASC

(ABnova, H00029108-P01) or NLRP-3 (Abnova, H00114548-P01) in binding buffer (100

mM Tris, 500 mM NaCl and 10mM DTT, pH 7). For the band shift competition assay, 0.5

1 tg of rabbit anti-ASC (Santa Cruz, sc-22514-R,) or 0.5 tg rabbit anti-NLRP3 (Cryo-2,

Adipogen) antibodies were used. Binding reactions were incubated at 15°C for 30 min,

loaded 454 onto a 6% non-denaturing, non-reducing polyacrylamide gel and ran in a 50

mM Tris (pH 7), 0.38 M glycine, and 2 mM EDTA buffer at 100 V for 2-3 hours. Mouse

IgG2A isotype control (R&D Systems, MAB03) was used as negative control antibody.

Mouse IgG2A isotype control, bovine actin (Sigma, A3653) and recombinant MMP-7

(Merck Millipore, #444270) (0.25 g each) were used as control for unspecific protein

binding. Gels were imaged using the Bio-RAD ChemiDocTM system.

Experimental urinary tractinfection

Mice were bred and housed in the specific pathogen-free MIG animal facilities

(Lund, Sweden) with free access to food and water. Female C57BL/6 mice or Illb+ (Horai

et al. Journal of Experimental medicine 1998:187(9):1463-75), Nlrp3+, Asc+ ( Mariathasan

S, etal. Nature. 2006;440(7081):228-32), Tlr4+ (Hoshino K, etal.JImmunol.

1999;162(7):3749-52), Caspl(Mariathasan et al. Nature 2004:430(6996):213-218), Irf3

(Sato M, etal. Immunity. 2000;13(4):539-48) orMmp74 (Wilson etal. Proc. NatlAcad Sci

USA 1997: 94(4): 1402-1407) mice were used at 9-15 weeks of age. The Illfb+ mice have

recently been shown to be functionally defective for IL-la (Freigang et al. Nat. Immunol.

2013;14(10):1045-1053). The Casp-1 mice were also deficient for Caspase-II (Kayagaki et

al. Nature 2011, 479 (7371:117-121).

Mice under Isofluorane anesthesia were intravesically infected (108 CFU in 0.1

ml) through a soft polyethylene catheter (outer diameter 0.61 mm; Clay Adams).

Animals were sacrificed under anesthesia; kidneys and bladders were asceptically

removed and in the case of bladders, macroscopic pathology was documented by

photography. Tissues were fixed with 4% paraformaldehyde or frozen for sectioning

and RNA extraction. Viable counts in homogenized tissues (Stomacher 80, Seward

Medical) were determined on TSA (37°C, overnight). Urine samples were collected prior

to and at regular times after infection and quantitatively cultured. Neutrophils in

uncentrifuged urine were counted, using a hemocytometer.

Histology and immunohistochemistry Tissues were embedded in OCT and 5- tm-thick fresh cryosections on positively

charged microscope slides (Superfrost/Plus; Thermo Fisher Scientific) were fixed with

4% paraformaldehyde or acetone-methanol (1:1). For hematoxylin/eosin or

immunohistochemistry, sections were blocked and permeabilized (0.2% Triton X-100, 5%

goat normal serum (DAKO) or 1% BSA (Sigma), stained (anti-neutrophil antibody

[NIMP-R14] (ab2557, Abcam), polyclonal E. coli antibody (1:100, NB200-579 Novus

Biologicals), rabbit anti-IL-1 beta (1:50, ab9722), anti-MMP-7 (1:100, ab4044), anti-ASC

(TMS1) (1:100, ab155970, all Abcam) or anti-NLRP3 (1:40, sc66846 Santacruz) rabbit

antibodies. Alexa 488 anti-rat IgG or anti-rabbit IgG and Alexa 568 anti-rabbit IgG were

secondary antibodies and nuclei were counterstained with DAPI (0.05 mM). Imaging

was by fluorescence microscopy (AX60, Olympus Optical). Richard -Allan Scientific

Signature Series Hemotoxylin 7211 and Eosin-Y 7111(Thermo Scientific) were used to

counterstain the tissue sections.

Ilib and Mmp7 therapy

The IL-1 receptor antagonist, Anakinra (Kineret©, SOBI,) or the broad-spectrum

MMP inhibitor, Batimastat (ab142087, Abcam) were injected i.p.

Statistics

P-values < 0.05 were considered significant. Prism version 6.02 (GraphPad),

Mann-Withney test (two tailed), One-way Anova (Kurkall-Wallis) and Two-way Anova

non-matching test were used as appropriate. Example 2

Acute cystitis strains elicit an IL-1 V response in human bladder epithelial cells

A mucosal cytokine response accompanies UTI and the epithelial cytokine

repertoire includes IL-Ip (Hedges et al. International Journal of antimicrobial agents 1994;

4(2): 89-93). A functional role of IL-Ip has not been characterized, however. To address

this question, human bladder epithelial cells (HTB-9) were infected in vitro with E. coli

strains causing acute cystitis. The uropathogenic E. coli (UPEC) strain CFT073 and the

asymptomatic bacteriuria strain E. coli 83972 (ABU) were used as controls. Cells were infected with 108CFU/ml for 4 hours with gentamicin added after 1 hour, to focus on the

early host response. Supernatants were collected for analysis in which inflammatory

mediators in cell supernatants were quantified.

A rapid IL-Ip response, indicated by IL-Ip secretion was detected following

infection with the acute cystitis (CY) strains CY-17, CY-92 and CY-132. E. coli CFT073

also triggered IL-Ip secretion. In contrast, the response to the ABU strain E. coli 83972

was low (P < 0.005 compared to uninfected cells), indicating a possible virulence

association (Figure 1A). Pro-IL-1p and mature IL-Ip were detected by Western blot

analysis of the supernatants (Fig 1B), suggesting that the acute cystitis strains activate de

novo IL-Ip synthesis and IL-Ip processing. The IL-Ip response was also shown to differ

among the CY strains tested, with a low response to CY-49 (Fig 1A and 1B).

To address if IL-ip activation is a characteristic of acute cystitis strains, we

infected human bladder epithelial cells with an epidemiologically defined collection of

paediatric acute cystitis isolates (n = 67). The majority of these strains (85 %) triggered an

IL-Ip response > 5 pg/ml and 64 % of those triggered a high response (40 - 1,000 pg/ml,

Fig IC). In contrast, a collection of paediatric ABU strains (n = 66), obtained by screening

of infants and children in the same geographic area and background population as the

CY strains, did not trigger a strong IL-Ip response (61 % < 5 pg/ml), resulting in

significantly higher mean IL-Ip concentrations for the CY than the ABU strains (121.8 and 32.4 pg/ml respectively, P < 0.001, Fig IC). The results suggest that the majority of acute cystitis strains activate an IL-ip response in human bladder epithelial cells.

As pro-IL-1p is processed by the inflammasome, we examined if infection

triggers an inflammasome response in human bladder epithelial cells. By confocal

microscopy, we visualized the infection-induced changes in ASC (Apoptosis-associated

speck-like protein containing a CARD), NLRP-3 (NACHT, LRR and PYD domains

containing protein 3), Caspase-1 and IL-ip levels (Fig ID). Inflammasome protein levels

increased after infection with CY-92, CY-17, CY-132 and CFT073 but the ABU strain had

no effect, consistent with the low IL-p.

The results suggest that in addition to the IL-p response, acute cystitis strains

stimulate an inflammasome response in human bladder epithelial cells..

Example 3

In vivo control of acute cystitis by I11b in the murine UTI model

The development of acute cystitis in mice after experimental infection with IL-p

inducing E. coli strains was examined. C57BL/6 WT mice were infected by intravesical

inoculation with acute cystitis strains that triggered high IL-Ip responses in human

bladder epithelial cells, in vitro (CY-17 or CY-92) or with CFT073. Bladder infection was

evaluated at sacrifice after 7 days (Fig 2A) and the kinetics of infection were followed in

urine samples obtained after 6 and 24 hours, 3 and 7 days (Fig 2B).

Infected bladders showed macroscopic evidence of moderate inflammation, with

an increase in size, edema and hyperaemia. Tissue pathology was confirmed by histology, with a loss of structure in infected mice, compared to uninfected controls (Fig

2A). Tissue inflammation was accompanied by an increase in urine neutrophil numbers

and bacterial numbers reached a peak after 24 hours and then declined (Fig 2B). Bacteria

and neutrophils were detected by immunohistochemistry in the bladder mucosa of

infected mice (Fig 2A) and a rapid IL-Ip response, as confirmed by mucosal staining for

IL-ip after 24 hours and elevated IL-ip levels in urine.

The role of IL-ip for the inflammatory response was then addressed, by infecting

Illb-/- mice with CFT073. The Illb-/- mice were protected from bladder pathology, had

lower bacterial counts and fewer infiltrating neutrophils than the C57BL/6 WT mice (Fig

2C, P < 0.001). Bladder morphology was intact and immunohistochemistry confirmed the low bacterial counts and the near absence of inflammatory cells in the tissues (Fig 2D).

The results demonstrate that clinical CY isolates cause acute cystitis in the mouse,

characterized by a self- limiting inflammatory response and controlled by IL-p.

Example 4

Hyper-activation of IL-1V in mice lacking ASC or NLRP-3

To inactivate the inflammasome-dependent processing of IL-ip, subsequently

infected NLRP-3 (Nlrp3+), ASC (Asce) or Caspase-1 (Casp1+) deficient mice, as described

above. C57BL/6 WT mice were included as controls. Major, genotype-specific differences in bladder pathology were detected after seven days (Fig 2C and 2D). Bladders from

Asc' and Nlrp3--mice were severely inflamed and enlarged with edema, hyperaemia

and thickened bladder walls. By histology, most bladders from Asc- mice showed

extensive loss of tissue structure with round cell infiltration and hypertrophy of the

bladder epithelium (10/14 mice, 71 %). Similar tissue destruction was observed in

bladders from Nlrp3+mice (7/11 mice, 169 64 %), (Fig 2D).

Bladder pathology was accompanied by high bacterial 170 counts in urine and

bladder tissue (Fig 2C). In Asce mice, the neutrophil influx accelerated until day 7,

indicating a loss of homeostatic control and progression to chronic inflammation.

Infiltrating bacteria and neutrophil aggregates or micro-abscesses were detected in the

mucosa of Asc' and Nlrp34 mice, with extensive sloughing of epithelial cells into the

lumen (Fig 2D). Infection was accompanied by strong epithelial IL-Ip staining in bladder

tissue sections after 24 hours and elevated urine IL-p levels. This hyper-inflammatory

phenotype was recreated in Asc+ mice after infection with the acute cystitis strains CY-92

and CY-17, which triggered high IL-1p responses in vitro.

Example 5

Tissue retention of IL-1p and bacterial persistence in Casp1-/- mice

In contrast, to Asc and Nlrp3+ mice, Casp14 mice were persistently infected but

did not develop acute bladder pathology. Neutrophils were not retained in bladder

tissues but were elevated in urine, suggesting that Casp1 mice fail to generate an

environment suitable for tissue neutrophil retention. Furthermore, bacteria were present

in urine but were not detected in the tissue sections. Accumulation of IL-p Casp1 mice

and low urine IL-1P confirmed the importance of Caspase-1 for IL-P secretion (Fig 4F).

The elevated bacterial numbers in Casp1+- Asc- and Nlrp3- mice, compared to WT mice

suggested that a functional inflammasome is essential for bacterial clearance from

infected bladders.

These studies are the first to reproduce essential aspects of acute cystitis in an

animal model and to define acute cystitis as an IL-1p-dependent inflammatory state.

Importantly, mice lacking functional ASC or NLRP-3 developed a hyper-inflammatory

state with extensive tissue damage. This gives rise to the therapeutic use of these

proteins in the prevention or treatment of cystitis. Example 6

Gene expression in infected bladders

To further characterize the molecular basis of bladder pathology, whole bladder

RNA was subjected to genome-wide transcriptomic analysis. Regulated genes were

defined in comparison with RNA from uninfected bladders of each genetic background

(fold change (FC) > 1.41, and P < 0.05, Figure 3).

About 1800 genes were altered exclusively in mice with bladder pathology (heat

map in Figure 3A, histogram in Figure 3B and Venn diagram in Figure 3C). Genes with

a FC > 100 included metalloproteinase Mmp7, the neutrophil chemoattractants Cxcl1 and

Cxcl6, the interferon-induced protein Ifit1 and calprotectine S100a8. By canonical

pathway analysis, significantly regulated genes were shown to control granulocyte and

leucocyte diapedesis and signaling, dendritic cell maturation, TNFR1 and 2-, NF-kB-, IL

6- and IL-10 signaling. These pathways were not significantly regulated in Illb or

control mice, suggesting a direct disease association.

To address the role of IL-Ip and the inflammasome for bladder pathology, genes

encoding inflammasome complex constituents, inflammasome activators or downstream

effectors were selected for analysis. A marked difference was observed, between mice

that developed acute cystitis and resistant mice (Figure 3D). Pathology was associated

with a drastic increase in overall gene expression in this family, and Cxcl1, Cxcl3, II b

and 1133 expression was most strongly regulated (FC 10-200). In contrast, 1118 and the

NLRP genes were not regulated. A weak response was observed for genes encoding ASC

(Asc) and Caspase-1. Innate immune regulators were moderately enhanced, such as the

NF-cB constituents, Myd88, Mapkl1 and chemokines Ccl-7, -5 and -2 (FC about 2).

Importantly, inflammasome gene expression was virtually absent in Illb+ mice further

emphasizing the key role of IL-ip for bladder pathology.

Networks of IL-ip dependent genes confirmed the over-activation of IL-ip

dependent gene expression in Asc' and Nlrp34 mice. In Illb mice, the IL-p dependent

signaling cascade was completely abrogated, suggesting a lack of alternative activation

mechanisms. Remaining, IL-ip -independent activated genes included the transcription

factor Irf3, which controls the innate immune response to kidney infection and is

activated via TLR4, TICAM-1 (TRIF) and TICAM-2 (TRAM) rather than the MyD88 and

NF-cB pathway (7). In addition, upregulation of genes related to the inflammasome

pathway was observed inIl b' mice (Cxcl3, Ccl12, Cc5, Naipi, Chuk), suggesting that

their activation is not dependent on IL-ip.

Example 7

Mechanism of atypical IL-lb processing

IL-ip processing was further examined in human bladder epithelial cells infected

with acute cystitis strains CY-17, CY-132 and CY-49, which generated the strongest IL-ip

response in the epidemiological survey (101 CFU/ml, 4h). CFT073 and ABU were

included as positive and negative controls.

Cell lysates and supernatants from infected cells were subjected to Western blot

analysis, with antibodies specific for IL-ip. Two fragments of approximately 29 kDa and

16-18 kDa were detected, corresponding to the N-terminal pro-piece and mature IL-ip.

In the presence of the Caspase-1 inhibitor Z-VAD, IL-ip processing was inhibited by

about 20%, suggesting that most of the IL-ip processing is caspase-independent.

Transcriptomic analysis was used to identify the most strongly upregulated

genes in mice with bladder pathology. The Mmp7 gene, which encodes the matrix

metalloproteinase (MMP-7) was identified as the most strongly regulated gene in mice

with pathology (FC > 200) as shown by gene expression and RT-PCR (Figure 4A and 4B).

High MMP-7 expression was also detected by immunohistochemistry in bladder tissue

sections from mice that developed pathology (Asc+ and Nlrp34 mice) (Fig 4C) where

staining was exclusively epithelial with shedding of MMP-7 positive epithelial celinto

the bladder lumen. Epithelial MMP-7 activation was detected as early as 24 hours after

infection but MMP-7 was not regulated in resistant mice (Il-b+or Casp1 mice) (Fig 4C) at7days. Increased IlIband Mmp7 mRNA levels were confirmed by RT-PCR, in these tissues compared to WT mice.

MMP-7 was subsequently shown to cleave or degrade IL-P, in vitro. GST-tagged

recombinant IL-1p was incubated with purified MMP-7 and proteolytic fragments were identified by Western blots using IL-1p specific antibodies (Figure 4D). Kinetic analysis

showed a time-dependent cleavage of IL-lb with a gradual reduction in full-length

protein from 10 to 60 minutes. After 24 hours, IL-Ip was completely degraded by MMP-7

(Fig 4D). Time-dependent ASC degradation by MMP-7 was also detected but NLRP-3

was not affected. The IL-Ip degradation products corresponded approximately to pro

IL-Ip (36kDa), the N-terminal fragment (29 kDa) and mature IL-Ip (16-18 kDa), also

seen in supernatant from cells infected with CY-92. Addition bands were also detected

after MMP-7 degradation (Fig 4D). NLRp-3 was not degraded and therefore serves as a

negative control for ASC degradation by MMP-7.

To further evaluate the involvement of MMP-7 in acute cystitis, Mmp7-/- mice

were infected with CFT073. No disease phenotype was detected (Fig 4E) and Illb and IL

1P- dependent gene expression was comparable to WT mice (Fig 3). Activated genes

were involved in IRFI-dependent IL-1 signaling, including Birc3, Myd88, Irfl and Cc5. A

moderate IL-Ip response was observed in these mice, with submucosal IL-Ip staining

and moderate mucosal accumulation (Fig 4F). IL-Ip levels in urine were comparable to

those of WT and Casp1+mice (Fig 4F).

The results identify an alternative MMP-7-dependent mechanism of pro-IL-p

processing in inflammasome-deficient Asc' and Nlrp34 mice.

Example 8

NLRP-3 and ASC act as negative regulators of MMP7 expression

To understand the mechanism of increased MMP-7 expression in infected Asc

and Nlrp34 mice, we examined if ASC and/or NLRP-3 may act as negative regulators of

MMP7 expression. ASC or NLRP-3 expression was inhibited by transfection of human

bladder epithelial cells with ASC- or NLRP3- specific siRNAs and the effects on MMP-7

expression were quantified by confocal imaging and Western blots. MMP-7 expression

increased drastically in transfected and infected cells where the expression of ASC or

NLRP-3 had been inhibited (Figs 5A and 5B). By co-immunoprecipitation, ASC was shown to pull down NLRP-3 in nuclear extracts of uninfected cells but after infection, a reduction in ASC/NLRP-3 interaction was detected suggesting that a loss of

ASC/NLRP-3 interaction in the nuclear compartment accompanies MMP7 activation.

To determine if ASC and NLRP-3 interact with the MMP7 promoter, DNA

fragments spanning the entire promoter were used as probes in electrophoretic mobility

shift assays (EMSA). A DNA fragment of 259 bp, adjacent to the transcription start site

(P1,position -18/-276) was shown to interact with a nuclear protein extract from infected

bladder cells, resulting in a significant band shift (Figs 5C and 5D). Specificity for ASC

and NLRP-3 was confirmed by competition with specific antibodies (Fig 5D). In the

absence of nuclear extract, the probe formed a single low molecular weight 256 band,

serving as a negative control. To confirm that ASC binds directly to theMMP7 promoter,

recombinant ASC protein was incubated with the 259 bp DNA sequence and examined

by EMSA. Strong dose-dependent binding of ASC to MMP7 promoter DNA was

detected as a band shift, which was competitively inhibited by specific antibodies (Fig

5E).

The results suggest that NLRP-3 and ASC act as negative regulators ofMMP7

expression and identify an ASC binding site inMMP7 promoter DNA, adjacent to the

transcription start site.

Example 9

Therapeutic intervention - efficacy of the IL-1 receptor antagonist and MMP inhibitor

The results suggested that an exaggerated IL-Ip response drives bladder

pathology. To address this hypothesis, Asc' mice were treated with the IL-1 receptor

antagonist Anakinra (IL-iRA), 30 minutes before infection and daily after infection with

E. coli CFT073 (1 mg in 100 tlof PBS i.p. per mouse for seven days, Figure 6A). A

dramatic therapeutic effect of Anakinra was observed compared to untreated Asc- mice

(Figure 6B-D). Anakinra-treated mice were protected against macroscopic bladder

pathology, edema and hyperemia (Figure 6, B and C). A marked reduction in pathology

was also observed in bladder tissue sections (Figure 6D). The mucosal neutrophil

infiltrate was prevented and mucosal integrity was maintained. Consistent with this

reduction in inflammation, urine neutrophil numbers were low (Figure 5E). Bacterial counts remained elevated however, suggesting a key role of IL-I signaling for bacterial clearance (Fig 6E).

To address the contribution of MMP-7 to pathology, Asc-/- mice were also treated

with an MMP inhibitor (Batimastat), (0.5 mg in 100tl of PBS i.p. 30 minutes before, first three and last three days of infection, Figure 6A). Treated mice were protected against

macroscopic bladder pathology (Figure 6B) with a reduction in the bladder edema and

hyperemia compared to untreated Asc-- mice (Figure 6C). Neutrophil infiltration was

markedly reduced (Figure 6D). By Western blot analysis of urine samples, mice treated

with IL-iRA showed a loss of mature IL-ip in urine and mice treated with the MMP

inhibitor showed defective processing of IL-1p. As in the IL-iRA-treated mice, bacterial

numbers remained elevated in MMPI treated mice (Fig 6E). By immunohistochemistry,

some retention of unprocessed IL-1p in bladder tissue was detected, consistent with the

need for MMP-7 and IL-iR to secrete mature IL-ip.

These results suggest that the hyper-inflammatory state that causes acute cystitis

can be attenuated by blocking the access of IL-1p to its receptor and by inhibiting

MMP-7. Thus these molecules are functional targets for immune-modulatory therapy. Example 10

IL-10 and MMP-7 responses in human bladder cells and patients with acute cystitis

To address if the IL-1p response of human bladder to infection with acute cystitis

strains is accompanied by an increase in MMP-7 protein levels, extracts of human

bladder cells were subjected to Western blot analysis. MMP-7 was detected in cells

infected with the acute cystitis strains or CFT073 but not with the ABU strain. The results

demonstrate that the human response to infection with acute cystitis strains includes

IL-1p /MMP-7.

To further address the human relevance of the IL-p/MMP-7 response, urine

samples were obtained from patient with acute cystitis at the time of diagnosis. A

diagnosis of acute cystitis was based on urgency, frequency and/or dysuria and a

positive dipstick but no fever (n=9). Samples were also obtained from patients with ABU

(n=31, who carried the prototype ABU strain E.coli 83972, following therapeutic

inoculation.

By ELISA, elevated concentrations of urine IL-1 (> 10 pg/ml) were detected in 9/9 patients with acute cystitis (Figure 7 A). The urine IL- response was significantly lower in patients with long-term ABU (P< 0.001). The urine samples were also examined for the MMP-7 content (Figure 7B. Samples positive for MMP-7 were found in both patient groups but the highest MMP-7 concentrations were present in the acute cystitis group (P < 0.001). The results show that patients with acute cystitis have elevated concentrations of IL-I1 and MMP-7 in urine, identifying IL-1 and MMP-7 as biomarkers of acute cystitis. Example 11 Different genes control the susceptibility to acute cystitis and acute pyelonephritis The clinical entities of acute pyelonephritis an acute cystitis typically are quite distinct, suggesting a difference in molecular pathogenesis. To test this hypothesis, we examined the kidneys from infected Asc- and Nlrp3-- mice. There was no evidence of macroscopic kidney pathology in the mice that developed severe acute cystitis and bacterial kidney counts were not elevated, compared to WT mice. Bacteria and scattered neutrophils were detected by immunohistochemistry, along the renal pelvic mucosa but the tissue structure was unaffected. As IrJ3 regulates the susceptibility to acute pyelonephritis, IrJ3 mice were used as positive controls for kidney pathology. After infection, their kidneys were severely inflamed, with macroscopic abscesses. Bacterial counts and neutrophil numbers were elevated compared to WT mice. Neutrophil infiltration and bacterial invasion was detected in the mucosa and collecting ducts. There was, however, no evidence of bladder pathology in IrJ3- mice. NLRP-3 and ASC staining resembled that in WT mice and the IL-1 response was weak. The results demonstrate that IL-1 and inflammasome genes do not control the susceptibility to acute pyelonephritis. Reference to any prior art in the specification is not an acknowledgement or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be combined with any other piece of prior art by a skilled person in the art. By way of clarification and for avoidance of doubt, as used herein and except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additions, components, integers or steps.

Claims (20)

Claims

1. A method for treating cystitis comprising administering to a patient in need

thereof, an effective amount of a reagent selected from the group consisting of IL-p

inhibitors and MMP inhibitors.

2. A method according to claim 1 wherein the cystitis is acute cystitis.

3. A method according to claim 1 wherein the reagent is an IL-1p inhibitor.

4. A method according to claim 3 wherein the IL-1p inhibitor is an interleukin-1

receptor antagonist

5. A method according to claim 4 wherein the IL-1p inhibitor is anakinra.

6. A method according to claim 1 wherein the reagent is an MMP inhibitor.

7. A method according to claim 6 wherein the MMP inhibitor is an MMP7 inhibitor.

8. A method according to claim 7 wherein the MMP7 inhibitor is Batimastat.

9. A reagent selected from the group consisting of IL-1p inhibitors and MMP

inhibitors for use in the treatment of cystitis.

10. A reagent according to claim 9 wherein the cystitis is acute cystitis.

11. A reagent according to claim 9 or claim 10 which is an IL-1p inhibitor.

12. A reagent according to claim I Iwherein the IL-ip inhibitor is an interleukin-i

receptor antagonist

13. A reagent according to claim 12 wherein the IL-1p inhibitor is anakinra.

14. A reagent according to claim 9 or claim 10 which is an MMP inhibitor.

15. A reagent according to claim 14 wherein the MMP inhibitor is an MMP7

inhibitor.

16. A reagent according to claim 15 wherein the MMP7 inhibitor is Batimastat.

17. A method for diagnosing cystitis and in particular, acute cystitis, said method

comprising detecting elevated levels of IL-1p or MMP-7 in urine of a subject.

18. A method for diagnosing susceptibility to cystitis, said method comprising

detecting a mutation in a gene encoding a protein selected from ASC or NLRP-3 which

results in downregulation of said gene and/or in the expression of inactive protein.

19. A method for preventing or treating cystitis in a patient susceptible thereto as a

result of a mutation which impacts on the expression of functional ASC or NLRP-3,

which method comprises administering to said patient a protein selected from ASC or

NLRP-3 or a functional fragment or variant thereof.

20. A protein selected from ASC or NLRP-3 or a functional fragment or variant thereof,

for use in the treatment of patients suffering from or susceptible to cystitis as a result of a

mutation which impacts on the expression of functional ASC or NLRP-3 respectively.